Abstract
Exogenous or endogenous beta(2)-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor beta1 (TGF-beta1), a critical mediator of acute lung injury, inhibits beta(2)-adrenergic receptor agonist-stimulated vectorial fluid and Cl(-) transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the beta(2)-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-beta type II receptor restored beta(2)-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-beta1 in impairing the beta- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.
Highlights
We verified our in vitro this second series of experiments demonstrates that transforming growth factor 1 (TGF-1) findings using an in vivo model of Acute lung injury (ALI) induced by hemorrhagic inhibits the 2AR agonist-stimulated CFTR-specific ClϪ transshock that we have shown to be associated with hyporespon- port across rat and human alveolar epithelia
The results showed that a 6-h exposure to TGF-1 significantly inhibited CFTR promoter activity and mRNA expression, an effect prevented by pretreatment with CPT-cAMP or phosphoinositide kinase (PIK)-90, a specific PI3K inhibitor (Fig. 6, E and F), but not by pretreatment with MAPK inhibitors
Experimental and clinical studies have shown that the cAMP-mediated stimulation of CFTR-dependent alveolar fluid clearance by endogenous or exogenous 2AR agonists is one of the major mechanisms that prevent the flooding of the airspaces after onset of ALI [5,6,7,8,9,10,11,12,13,14,15, 23]
Summary
Reagents—All of the cell culture media were prepared by the University of California, San Francisco Cell Culture Facility using deionized water and analytical grade reagents. The cells were incubated in Dulbecco’s modified Eagle’s medium containing magnetic beads coated with an anti-CD-14 antibody (Dynabeads M/450 CD14; Dynal, Oslo, Norway) at 4 °C for 40 min under constant mixing to eliminate macrophages. After freshly isolated rat ATII cells were plated on Transwells and grown in an air-liquid interface for 24 h, 150 l of medium containing 0.5 Ci/ml [125I]albumin was added to the apical side of the monolayers, and the Transwells were placed in a humidified tent. Saturation Binding Experiments—The plasma membraneenriched fractions (1 g of membrane protein) from primary rat alveolar type II cells were incubated with different concentrations (0 –160 pM) of the -AR antagonist [125I]ICYP for 90 min at 37 °C in the following binding buffer: 50 mM Tris1⁄7HCl, 100 mM KCl, and 5 mM MgCl2, pH 7.4. The reactions were stopped by dilution with 4 ml of ice-cold washing buffer
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